PROJECT SUMMARY/ABSTRACT During cell division chromosomes must be accurately segregated to produce daughter cells with the correct numbers of chromosomes whereas segregation errors generate aneuploid cells with abnormal numbers of chromosomes. In normal human somatic cells, chromosome segregation errors and aneuploidy are rare. In contrast, in human totipotent and pluripotent embryonic cells meiotic and mitotic errors are common, resulting in aneuploidy being the leading cause of miscarriages and birth defects. Yet, we do not understand the mechanisms responsible for this, particularly for mitotic errors. Moreover, given such a high mitotic error rate, it is proposed that diploid cells can outcompete aneuploid cells as development progresses to establish euploid embryos, but how this occurs is unknown. We will address these gaps in our knowledge using pluripotent human embryonic stem cells (hESCs) and a combination of quantitative imaging, chemical, and genomics approaches. The specific aims of this grant are (1) to determine the mitotic pathways responsible for chromosome segregation errors in hESCs, (2) to determine if the G1 cell cycle structure of hESCs permits an initial tolerance to aneuploidy, and (3) to determine how an aneuploid genome subsequently impairs hESCs contribution to embryonic tissues. Collectively, these aims test our overarching hypothesis that pluripotent embryonic cells are inherently different from somatic cells with respect to mechanisms that support chromosome segregation fidelity and in their response to aneuploidy. Furthermore, this work lays the foundation for our long-term objectives of identifying the molecular signaling pathways responsible for the causes, tolerance, and consequences of aneuploidy in embryonic cells and for developing strategies that preserve the genome integrity of embryonic cells grown in culture to improve the success of reproductive and regenerative medicine therapies. In conclusion, this work will reveal both the mechanisms underlying aneuploidy in embryonic cells and how karyotype stability is eventually achieved to support normal human development.